τ-conotoxin peptides

ABSTRACT

The invention relates to relatively short peptides (termed τ-conotoxins herein), about 10-25 residues in length, which are naturally available in minute amounts in the venom of the cone snails or analogous to the naturally available peptides, and which preferably include two disulfide bonds.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a continuation of U.S. patent applicationSer. No. 10/619,473, filed on 16 Jul. 2003, which in turn is a divisionof U.S. patent application Ser. No. 09/497,491, filed Feb. 4, 2000, nowU.S. Pat. No. 6,630,573. Ser. No. 09/497,491 claims benefit of andpriority under 35 U.S.C. § 119(e) to U.S. Provisional Patent ApplicationNo. 60/118,642, filed Feb. 4, 1999. The disclosures of the aboveidentified applications are hereby incorporated by reference.

This invention was made with Government support under Grant No. PO1GM48677 awarded by the National Institute of General Medical Sciences,National Institutes of Health, Bethesda, Md. The United StatesGovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

The invention relates to relatively short peptides (termed τ-conotoxinsherein), about 10-20 residues in length, which are naturally availablein minute amounts in the venom of the cone snails or analogous to thenaturally available peptides, and which preferably include two disulfidebonds.

The publications and other materials used herein to illuminate thebackground of the invention, and in particular, cases to provideadditional details respecting the practice, are incorporated byreference, and for convenience are referenced in the following text byauthor and date and are listed alphabetically by author in the appendedbibliography.

The predatory cone snails (Conus) have developed a unique biologicalstrategy. Their venom contains relatively small peptides that aretargeted to various neuromuscular receptors and may be equivalent intheir pharmacological diversity to the alkaloids of plants or secondarymetabolites of microorganisms. Many of these peptides are among thesmallest nucleic acid-encoded translation products having definedconformations, and as such, they are somewhat unusual. Peptides in thissize range normally equilibrate among many conformations. Proteinshaving a fixed conformation are generally much larger.

The cone snails that produce these peptides are a large genus ofvenomous gastropods comprising approximately 500 species. All cone snailspecies are predators that inject venom to capture prey, and thespectrum of animals that the genus as a whole can envenomate is broad. Awide variety of hunting strategies are used, however, every Conusspecies uses fundamentally the same basic pattern of envenomation.

Several peptides isolated from Conus venoms have been characterized.These include the α-, μ- and ω-conotoxins which target nicotinicacetylcholine receptors, muscle sodium channels, and neuronal calciumchannels, respectively (Olivera et al., 1985). Conopressins, which arevasopressin analogs, have also been identified (Cruz et al., 1987). Inaddition, peptides named conantokins have been isolated from Conusgeographus and Conus tulipa (Mena et al., 1990; Haack et al., 1990).

Chronic or intractable pain, which may result from degenerativeconditions or debilitating diseases, is currently treated with a varietyof analgesic compounds, often opioid compounds such as morphine.Likewise, neuropathic pain, typically a chronic condition attributableto injury or partial transection of a peripheral nerve, is alsoconventionally treated with opioid compounds such as morphine.

Conventional therapies for pain produce analgesia—a loss of sensitivityto pain without the loss of consciousness. Opioid compounds have beenused widely to produce analgesia, including plant-derived opioids suchas morphine, and endogenous opioids such as met- and leu-enkephalins, aswell as beta-endorphin.

Opioid compounds, while effective in producing analgesia for many typesof pain, may induce tolerance in some patients. When a patient becomestolerant, increasing doses of the opioid are required to produce thedesired analgesic effect. In addition, these compounds frequently resultin a physical dependence in patients, and may have side effects at highdoses.

The analgesic effects and adverse actions of various N-methyl-D-asparticacid (NMDA) receptor antagonists has been shown to vary depending on thesite of action and potency of the drug. For example, N-methyl-D-asparticacid (NMDA) receptor antagonists acting at the ion channel in anoncompetitive manner (e.g., MK-801 and phenylcyclidine (PCP)) orcompetitive inhibitors, show analgesic activity but show motorimpairment at equivalent doses. Glycine B-site N-methyl-D-aspartic acid(NMDA) antagonists appear to have analgesic activity at doses that donot impair motor function. Conantokins, which are polyamine-siteN-methyl-D-aspartic acid (NMDA) antagonist compounds have analgesiceffects at doses which do not produce overt side effects (PCT publishedapplication WO 98/03189).

It is desired to provide additional compounds which have analgesicproperties.

SUMMARY OF THE INVENTION

The invention relates to relatively short peptides (termed τ-conotoxinsherein), about 10-25 residues in length, which are naturally availablein minute amounts in the venom of the cone snails or analogous to thenaturally available peptides, and which preferably include two disulfidebonds.

More specifically, the present invention is directed to τ-conotoxinpeptides having the general formula I:

Xaa₁-Xaa₂-Xaa₃-Xaa₄-Cys-Cys-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Cys-Cys-Xaa₁₀-Xaa₁₁-Xaa₁₂-Xaa₁₃-Xaa₁₄-Xaa₁₅-Xaa₁₆-Xaa₁₇-Xaa₁₈-Xaa₁₉(SEQ ID NO:1), wherein Xaa₁ is des-Xaa₁, Asp, Glu or γ-carboxy-Glu(Gla); Xaa₂ is des-Xaa₂, Gln, Asn, Glu, Trp (D or L), neo-Trp, halo-Trpor any unnatural aromatic amino acid; Xaa₃ is des-Xaa₃, Gly, Ala, Asn orGln; Xaa₄ is des-Xaa₄, Val, Leu (D or L), Ile, Ala, Gly, Glu, Gla, Asp,Ser, Thr, Phe, Trp (D or L), neo-Trp, halo-Trp (D or L) or any unnaturalaromatic amino acid; Xaa₅ is Pro, hydroxy-Pro, Gln, Asn, Glu, Gla, Ala,Gly, Lys, Arg, Ile, Val, homoarginine, ornithine, N-methyl-Lys,N,N-dimethyl-Lys, N,N,N-trimethyl-Lys or any unnatural basic amino acid;Xaa₆ is Val, Phe, Thr, Ser, Glu, Gla, Asp, Asn, Gln, Ala, Gly, Ile, Leu(D or L) Met, Pro, hydroxy-Pro, Arg, homoarginine, ornithine, Lys,N-methyl-Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys, any unnatural basicamino acid or any unnatural aromatic amino acid; Xaa₇ is any Val, Ile,Asn, Leu (D or L), Gln, Gly, Ala, Phe, Glu, Gla, Arg, ornithine,homoarginine, Lys, N-methy-Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys,any unnatural basic amino acid or any unnatural aromatic amino acid;Xaa₈ is Ile, Leu (D or L), Met, Thr, Ser, Pro, hydroxy-Pro, Gln, Asp,Glu, Gla, Asn, Arg, homoarginine, ornithine, Lys, N-methy-Lys,N,N-dimethyl-Lys, N,N,N-trimethyl-Lys, Tyr, nor-Tyr, mono-halo-Tyr,di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr, any unnatural basicamino acid, any unnatural aromatic amino acid or any unnatural hydroxycontaining amino acid; Xaa₉ is des-Xaa₉, Ala, Gly, Asp, Glu, Gla, Trp (Dor L) neo-Trp, halo-Trp (D or L), Lys, N-methy-Lys, N,N-dimethyl-Lys,N,N,N-trimethyl-Lys, Arg, homoarginine, ornithine, Tyr, nor-Tyr,mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr orany unnatural basic amino acid; Xaa₁₀ is des-Xaa₁₀, Ile, Leu (D or L),Val, Glu, Gla, Asp, Thr, Ser, Pro, hydroxy-Pro, Trp (D or L), neo-Trp,halo-Trp (D or L), Phe, any unnatural aromatic amino acid or anyunnatural hydroxy containing amino acid; Xaa₁₁ is des-Xaa₁₁, Gln, Asn,Leu (D or L), Ile, Val, Ala, Gly, Trp (D or L), neo-Trp, halo-Trp (D orL), Arg, homoarginine, ornithine, Lys, N-methy-Lys, N,N-dimethyl-Lys,N,N,N-trimethyl-Lys, any unnatural basic amino acid or any unnaturalaromatic amino acid; Xaa₁₂ is des-Xaa₁₂, Ala, Gly, Phe, Trp (D or L),neo-Trp, halo-Trp (D or L) or any unnatural aromatic amino acid; Xaa₁₃is des-Xaa₁₃, Glu, Gla, Asp, Phe or any unnatural aromatic amino acid;Xaa₁₄ is des-Xaa₁₄, Ile, Val or Leu (D or L); Xaa₁₅ is des-Xaa₁₅, Thr,Ser, Ag, homoarginine, ornithine, Lys, N-methy-Lys, N,N-dimethyl-Lys,N,N,N-trimethyl-Lys or any unnatural basic amino acid; Xaa₁₆ isdes-Xaa₁₆, Glu, Gla or Asp; Xaa₁₇ is des-Xaa₁₇, Asn or Gln; Xaa₁₈ isdes-Xaa₁₈, Asp, Glu or Gla; Xaa₁₉ is des-Xaa₁₉, Phe or any unnaturalaromatic amino acid. The C-terminus may contain a free carboxyl group oran amide group. The halo is preferably bromine, chlorine or iodine, morepreferably iodine for Tyr and bromine for Trp. The Cys residues may bein D or L configuration and may optionally be substituted withhomocysteine (D or L). The Tyr residues may be substituted with the3-hydroxyl or 2-hydroxyl isomers and corresponding O-sulpho- andO-phospho-derivatives. The acidic amino acid residues may be substitutedwith any synthetic acidic amino acid, e.g., tetrazolyl derivatives ofGly and Ala.

The present invention is also directed to novel specific τ-conotoxinpeptides of general formula I having the formulas:

Phe-Cys-Cys-Xaa₁-Val-Ile-Arg-Xaa₂- (SEQ ID NO:2) Cys-Cys-Xaa₃;Phe-Cys-Cys-Xaa₁-Phe-Ile-Arg-Xaa₂- (SEQ ID NO:3) Cys-Cys-Xaa₃;Cys-Cys-Gln-Thr-Phe-Xaa₂-Xaa₃-Cys- (SEQ ID NO:4) Cys-Gln;Xaa₄-Gly-Xaa₃-Cys-Cys-Xaa₅-Xaa₆-Asn- (SEQ ID NO:5) Ile-Ala-Cys-Cys-Ile;Gly-Cys-Cys-Ala-Arg-Leu-Thr-Cys-Cys- (SEQ ID NO:6) Val;Asn-Gly-Cys-Cys-Xaa₁-Xaa₅-Gln-Met- (SEQ ID NO:7) Arg-Cys-Cys-Thr;Asp-Xaa₃-Asn-Ser-Cys-Cys-Gly-Xaa₅- (SEQ ID NO:8)Asn-Xaa₁-Gly-Cys-Cys-Xaa₁-Xaa₃; Xaa₄-Gly-Xaa₃-Cys-Cys-Xaa₅-Xaa₆-Asn-(SEQ ID NO:9) Ile-Arg-Cys-Cys-Val; Xaa₆-Cys-Cys-Xaa₆-Asp-Gly-Xaa₃-Cys-(SEQ ID NO:10) Cys-Thr-Ala-Ala-Xaa₁-Leu-Thr;Gly-Cys-Cys-Xaa₆-Asp-Gly-Xaa₃-Cys- (SEQ ID NO:11)Cys-Thr-Ala-Ala-Xaa₁-Leu-Thr; Asn-Gly-Cys-Cys-Arg-Ala-Gly-Asp-Cys- (SEQID NO:12) Cys-Ser-Arg-Phe-Xaa₆-Ile-Xaa₅-Xaa₆- Asn-Asp-Phe;Asn-Ala-Cys-Cys-Ile-Val-Arg-Gln-Cys- (SEQ ID NO:13) Cys;Asn-Gly-Cys-Cys-Arg-Ala-Gly-Asp-Cys- (SEQ ID NO:14) Cys-Ser;Cys-Cys-Xaa₁-Arg-Arg-Leu-Ala-Cys- (SEQ ID NO:15) Cys-Ile-Ile;Cys-Cys-Xaa₁-Asn-Xaa₅-Xaa₁-Cys-Cys- (SEQ ID NO:16) Phe-Ile;Gly-Cys-Cys-Ala-Met-Leu-Thr-Cys-Cys- (SEQ ID NO:17) Val;Leu-Cys-Cys-Val-Thr-Xaa₆-Asp-Xaa₃- (SEQ ID NO:18)Cys-Cys-Xaa₆-Xaa₃-Xaa₃; and Val-Cys-Cys-Arg-Xaa₁-Val-Gln-Asp- (SEQ IDNO:19) Cys-Cys-Ser;wherein Xaa₁ is Pro or hydroxy-Pro; Xaa₂ is Tyr, mono-halo-Tyr,di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr or nitro-Tyr; Xaa₃ is Trp orhalo-Trp; Xaa₄ is Gln or pyro-Glu; Xaa₅ is Lys, N-methyl-Lys,N,N-dimethyl-Lys or N,n,N-trimethyl-Lys, Xaa₆ is Glu orgamma-carboxy-Glu (Gla); and the C-terminus contains a carboxyl or amidegroup. The halo is preferably bromine, chlorine or iodine, morepreferably iodine for Tyr and bromine for Trp. In addition, the Argresidues may be substituted by Lys, ornithine, homoargine, N-methyl-Lys,N,N-dimethyl-Lys, N,N,N-trimethyl-Lys or any unnatural basic amino acid;the Lys residues may be substituted by Arg, ornithine, homoargine,N-methyl-Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys or any unnaturalbasic amino acid; the Tyr residues may be substituted with any unnaturalhydroxy containing amino acid; the Ser residues may be substituted withThr; the Thr residues may be substituted with Ser; and the Phe and Trpresidues may be substituted with any unnatural aromatic amino acid. TheCys residues may be in D or L configuration and may optionally besubstituted with homocysteine (D or L). The Tyr residues maybesubstituted with the 3-hydroxyl or 2-hydroxyl isomers and correspondingO-sulpho- and O-phospho-derivatives. The acidic amino acid residues maybe substituted with any synthetic acidic amino acid, e.g., tetrazolylderivatives of Gly and Ala.

More specifically, the present invention is directed to the followingτ-conotoxin peptides of general formula I:

AuVA: SEQ ID NO:2, wherein Xaa₁ is Pro, Xaa₂ is Tyr and Xaa₃ is Trp;AuVB: SEQ ID NO:3, wherein Xaa₁ is Pro, Xaa₂ is Tyr and Xaa₃ is Trp;Tx5.1: SEQ ID NO:4, wherein Xaa₂ is Tyr and Xaa₃ is Trp; G5.1: SEQ IDNO:5, wherein Xaa₃ is Trp, Xaa₄ is Gln, Xaa₅ is Lys and Xaa₆ is Glu;Qc5.1: SEQ ID NO:6; PVA: SEQ ID NO:7, wherein Xaa₁ is Pro and Xaa₅ isLys; Im5.1: SEQ ID NO:8, wherein Xaa₁ is Pro, Xaa₃ is Trp and Xaa₅ isLys; G5.2: SEQ ID NO:9, wherein Xaa₃ is Trp, Xaa₄ is Gln, Xaa₅ is Lysand Xaa₆ is Glu; Tx5.2a: SEQ ID NO:10, wherein Xaa₁ is Pro, Xaa₃ is Trpand Xaa₆ is Glu; Tx5.2b: SEQ ID NO:11, wherein Xaa₁ is Pro, Xaa₃ is Trpand Xaa₆ is Glu; Mr5.1: SEQ ID NO:12, wherein Xaa₅ is Lys and Xaa₆ isGlu; Mr5.2: SEQ ID NO:13; Mr5.3: SEQ ID NO:14; Ca5.1: SEQ ID NO:15,wherein Xaa₁ is Pro; Ca5.2: SEQ ID NO:16, wherein Xaa₁ is Pro and Xaa₅is Lys; Qc5.2: SEQ ID NO:17; Gm5.1: SEQ ID NO:18, wherein Xaa₃ is Trpand Xaa₆ is Glu; and Gm5.2: SEQ ID NO:19, wherein Xaa₁ is Pro.The C-terminus preferably contains a carboxyl group for the peptidesAuVA, AuVB, G5.1, PVA, G5.2, Mr5.2, Mr5.3 and Gm5.1 The C-terminus ofthe other peptides preferably contains an amide group.

Examples of unnatural aromatic amino acid include, but are not limitedto, such as nitro-Phe, 4-substituted-Phe wherein the substituent isC₁-C₃ alkyl, carboxyl, hydroxymethyl, sulphomethyl, halo, phenyl, —CHO,—CN, —SO₃H and —NHAc. Examples of unnatural hydroxy containing aminoacid, include, but are not limited to, such as 4-hydroxymethyl-Phe,4-hydroxyphenyl-Gly, 2,6-dimethyl-Tyr and 5-amino-Tyr. Examples ofunnatural basic amino acids include, but are not limited to,N-1-(2-pyrazolinyl)-Arg, 2-(4-piperinyl)-Gly, 2-(4-piperinyl)-Ala,2-[3-(2S)pyrrolininyl)-Gly and 2-[3-(2S)pyrrolininyl)-Ala. These andother unnatural basic amino acids, unnatural hydroxy containing aminoacids or unnatural aromatic amino acids are described in Building BlockIndex, Version 3.0 (1999 Catalog, pages 4-47 for hydroxy containingamino acids and aromatic amino acids and pages 66-87 for basic aminoacids; see also http://www.amino-acids.com), incorporated herein byreference, by and available from RSP Amino Acid Analogues, Inc.,Worcester, Mass. Examples of synthetic acid amino acids include thosederivatives bearing acidic functionality, including carboxyl, phosphate,sulfonate and synthetic tetrazolyl derivatives such as described byOrnstein et al. (1993) and in U.S. Pat. No. 5,331,001, each incorporatedherein by reference.

Optionally, in the peptides of general formula I and the specificpeptides described above, the Asn residues may be modified to contain anN-glycan and the Ser and Thr residues may be modified to contain anO-glycan. In accordance with the present invention, a glycan shall meanany N—, S— or O-linked mono-, di-, tri-, poly- or oligosaccharide thatcan be attached to any hydroxy, amino or thiol group of natural ormodified amino acids by synthetic or enzymatic methodologies known inthe art. The monosaccharides making up the glycan can include D-allose,D-altrose, D-glucose, D-mannose, D-gulose, D-idose, D-galactose,D-talose, D-galactosamine, D-glucosamine, D-N-acetyl-glucosamine(GlcNAc), D-N-acetyl-galactosamine (GalNAc), D-fucose or D-arabinose.These saccharides may be structurally modified, e.g., with one or moreO-sulfate, O-phosphate, O-acetyl or acidic groups, such as sialic acid,including combinations thereof. The gylcan may also include similarpolyhydroxy groups, such as D-penicillamine 2,5 and halogenatedderivatives thereof or polypropylene glycol derivatives. The glycosidiclinkage is beta and 1-4 or 1-3, preferably 1-3. The linkage between theglycan and the amino acid may be alpha or beta, preferably alpha and is1-.

Core O-glycans have been described by Van de Steen et al. (1998),incorporated herein by reference. Mucin type O-linked oligosaccharidesare attached to Ser or Thr (or other hydroxylated residues of thepresent peptides) by a GalNAc residue. The monosaccharide buildingblocks and the linkage attached to this first GalNAc residue define the“core glycans,” of which eight have been identified. The type ofglycosidic linkage (orientation and connectivities) are defined for eachcore glycan. Suitable glycans and glycan analogs are described furtherin U.S. Ser. No. 09/420,797, filed 19 Oct. 1999 and in PCT ApplicationNo. PCT/US99/24380, filed 19 Oct. 1999, both incorporated herein byreference. A preferred glycan is Gal(β1→3)GalNAc(α1→).

Optionally, in the peptides of general formulas I and II and thespecific peptides described above, pairs of Cys residues may be replacedpairwise with isoteric lactam or ester-thioether replacements, such asSer/(Glu or Asp), Lys/(Glu or Asp) or Cys/Ala combinations. Sequentialcoupling by known methods (Barnay et al., 2000; Hruby et al., 1994;Bitan et al., 1997) allows replacement of native Cys bridges with lactambridges. Thioether analogs may be readily synthesized using halo-Alaresidues commercially available from RSP Amino Acid Analogues.

The present invention is further directed to propeptides and nucleicacid sequences encoding the propeptides or peptides as described infurther detail herein.

SUMMARY OF THE SEQUENCE LISTING

SEQ ID NO:1 is generic formula I for τ-conotoxin peptides. SEQ ID NO:2is a generic formula for the peptide AuVA. SEQ ID NO:3 is a genericformula for the peptide AuVB. SEQ ID NO:4 is a generic formula for thepeptide Tx5.1. SEQ ID NO:5 is a generic formula for the peptide G5.1.SEQ ID NO:6 is a generic formula for the peptide Qc5.1. SEQ ID NO:7 is ageneric formula for the peptide PVA. SEQ ID NO:8 is a generic formulafor the peptide Im5.1. SEQ ID NO:9 is a generic sequence for the peptideG5.2. SEQ ID NO:10 is a generic sequence for the peptide Tx5.2a. SEQ IDNO:11 is a generic sequence for the peptide Tx5.2b. SEQ ID NO:12 is ageneric sequence for the peptide Mr5.1. SEQ ID NO:13 is a genericsequence for the peptide Mr5.2. SEQ ID NO:14 is a generic formula forthe peptide Mr5.3. SEQ ID NO:15 is a generic formula for the peptideCa5.1. SEQ ID NO:16 is a generic formula for the peptide Ca5.2. SEQ IDNO:17 is a generic formula for the peptide Qc5.2. SEQ ID NO:18 is ageneric formula for the peptide Gm5.1. SEQ ID NO:19 is a generic formulafor the peptide Gm5.2. SEQ ID NO:20 is a DNA sequence coding for theTx5.1 propeptide. SEQ ID NO:21 is the amino acid sequence of the Tx5.1propeptide. SEQ ID NO:22 is a DNA sequence coding for the G5.1propeptide. SEQ ID NO:23 is the amino acid sequence of the G5.1propeptide. SEQ ID NO:24 is a DNA sequence coding for the Qc5.1propeptide. SEQ ID NO:25 is the amino acid sequence of the Qc5.1propeptide. SEQ ID NO:26 is a DNA sequence coding for the Im5.1propeptide. SEQ ID NO:27 is the amino acid sequence of the Im5.1propeptide. SEQ ID NO:28 is a DNA sequence coding for the G5.2propeptide. SEQ ID NO:29 is the amino acid sequence of the G5.2propeptide. SEQ ID NO:30 is a DNA sequence coding for the Tx5.2propeptide. SEQ ID NO:31 is the amino acid sequence of the Tx5.2propeptide. SEQ ID NO:32 is a DNA sequence coding for the Tx5.3propeptide. SEQ ID NO:33 is the amino acid sequence of the Tx5.3propeptide. SEQ ID NO:34 is a DNA sequence coding for the Mr5.1 peptide.SEQ ID NO:35 is the amino acid sequence of the Mr5.1 peptide. SEQ IDNO:36 is a DNA sequence coding for the Mr5.2 peptide. SEQ ID NO:37 isthe amino acid sequence of the Mr5.2 peptide. SEQ ID NO:38 is a DNAsequence coding for the Mr5.3 propeptide. SEQ ID NO:39 is the amino acidsequence of the Mr5.3 propeptide. SEQ ID NO:40 is a DNA sequence codingfor the Ca5.1 propeptide. SEQ ID NO:41 is the amino acid sequence of theCa5.1 propeptide. SEQ ID NO:42 is a DNA sequence coding for the Ca5.2propeptide. SEQ ID NO:43 is the amino acid sequence of the Ca5.2propeptide. SEQ ID NO:44 is a DNA sequence coding for the Qc5.2propeptide. SEQ ID NO:45 is the amino acid sequence of the Qc5.2propeptide. SEQ ID NO:46 is a DNA sequence coding for the Gm5.1propeptide. SEQ ID NO:47 is the amino acid sequence of the Gm5.1propeptide. SEQ ID NO:48 is a DNA sequence coding for the Gm5.2propeptide. SEQ ID NO:49 is the amino acid sequence of the Gm5.2propeptide.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The invention relates to relatively short peptides (termed τ-conotoxinsherein), about 10-25 residues in length, which are naturally availablein minute amounts in the venom of the cone snails or analogous to thenaturally available peptides, and which preferably include two disulfidebonds.

The present invention, in another aspect, relates to a pharmaceuticalcomposition comprising an effective amount of an τ-conotoxin peptide, amutein thereof, an analog thereof, an active fragment thereof orpharmaceutically acceptable salts. Such a pharmaceutical composition hasthe capability of acting as an antagonist for acetylcholine receptorsand as analgesic agents for the treatment of pain, including migraine.Thus, the pharmaceutical compositions of the present invention areuseful in the treatment of pain (whether acute or chronic), includingchronic pain, and neuropathic pain, without undesirable side effects.

The τ-conotoxin peptides described herein are sufficiently small to bechemically synthesized. General chemical syntheses for preparing theforegoing τ-conotoxin peptides are described hereinafter. Various onesof the τ-conotoxin peptides can also be obtained by isolation andpurification from specific Conus species using the technique describedin U.S. Pat. No. 4,447,356 (Olivera et al., 1984); U.S. Pat. Nos.5,514,774; 5,719,264; and 5,591,821, as well as in PCT publishedapplication WO 98/03189, the disclosures of which are incorporatedherein by reference.

Although the τ-conotoxin peptides of the present invention can beobtained by purification from cone snails, because the amounts ofτ-conotoxin peptides obtainable from individual snails are very small,the desired substantially pure τ-conotoxin peptides are best practicallyobtained in commercially valuable amounts by chemical synthesis usingsolid-phase strategy. For example, the yield from a single cone snailmay be about 10 micrograms or less of τ-conotoxin peptide. By“substantially pure” is meant that the peptide is present in thesubstantial absence of other biological molecules of the same type; itis preferably present in an amount of at least about 85% purity andpreferably at least about 95% purity. Chemical synthesis of biologicallyactive τ-conotoxin peptides depends of course upon correct determinationof the amino acid sequence.

The τ-conotoxin peptides can also be produced by recombinant DNAtechniques well known in the art. Such techniques are described bySambrook et al. (1989). A gene of interest (i.e., a gene that encodes asuitable τ-conotoxin peptide) can be inserted into a cloning site of asuitable expression vector by using standard techniques. Thesetechniques are well known to those skilled in the art. The expressionvector containing the gene of interest may then be used to transfect thedesired cell line. Standard transfection techniques such as calciumphosphate co-precipitation, DEAE-dextran transfection or electroporationmay be utilized. A wide variety of host/expression vector combinationsmay be used to express a gene encoding a conotoxin peptide of interest.Such combinations are well known to a skilled artisan. The peptidesproduced in this manner are isolated, reduced if necessary, and oxidizedto from the correct disulfide bonds.

One method of forming disulfide bonds in the τ-conotoxin peptides of thepresent invention is the air oxidation of the linear peptides forprolonged periods under cold room temperatures or at room temperature.This procedure results in the creation of a substantial amount of thebioactive, disulfide-linked peptides. The oxidized peptides arefractionated using reverse-phase high performance liquid chromatography(HPLC) or the like, to separate peptides having different linkedconfigurations. Thereafter, either by comparing these fractions with theelution of the native material or by using a simple assay, theparticular fraction having the correct linkage for maximum biologicalpotency is easily determined. However, because of the dilution resultingfrom the presence of other fractions of less biopotency, a somewhathigher dosage may be required.

The peptides are synthesized by a suitable method, such as byexclusively solid-phase techniques, by partial solid-phase techniques,by fragment condensation or by classical solution couplings.

In conventional solution phase peptide synthesis, the peptide chain canbe prepared by a series of coupling reactions in which constituent aminoacids are added to the growing peptide chain in the desired sequence.Use of various coupling reagents, e.g., dicyclohexylcarbodiimide ordiisopropylcarbonyldimidazole, various active esters, e.g., esters ofN-hydroxyphthalimide or N-hydroxy-succinimide, and the various cleavagereagents, to carry out reaction in solution, with subsequent isolationand purification of intermediates, is well known classical peptidemethodology. Classical solution synthesis is described in detail in thetreatise, “Methoden der Organischen Chemie (Houben-Weyl): Synthese vonPeptiden,” (1974). Techniques of exclusively solid-phase synthesis areset forth in the textbook, “Solid-Phase Peptide Synthesis,” (Stewart andYoung, 1969), and are exemplified by the disclosure of U.S. Pat. No.4,105,603 (Vale et al., 1978). The fragment condensation method ofsynthesis is exemplified in U.S. Pat. No. 3,972,859 (1976). Otheravailable syntheses are exemplified by U.S. Pat. No. 3,842,067 (1974)and U.S. Pat. No. 3,862,925 (1975). The synthesis of peptides containingγ-carboxyglutamic acid residues is exemplified by Rivier et al. (1987),Nishiuchi et al. (1993) and Zhou et al. (1996).

Common to such chemical syntheses is the protection of the labile sidechain groups of the various amino acid moieties with suitable protectinggroups which will prevent a chemical reaction from occurring at thatsite until the group is ultimately removed. Usually also common is theprotection of an α-amino group on an amino acid or a fragment while thatentity reacts at the carboxyl group, followed by the selective removalof the α-amino protecting group to allow subsequent reaction to takeplace at that location. Accordingly, it is common that, as a step insuch a synthesis, an intermediate compound is produced which includeseach of the amino acid residues located in its desired sequence in thepeptide chain with appropriate side-chain protecting groups linked tovarious ones of the residues having labile side chains.

As far as the selection of a side chain amino protecting group isconcerned, generally one is chosen which is not removed duringdeprotection of the α-amino groups during the synthesis. However, forsome amino acids, e.g., His, protection is not generally necessary. Inselecting a particular side chain protecting group to be used in thesynthesis of the peptides, the following general rules are followed: (a)the protecting group preferably retains its protecting properties and isnot split off under coupling conditions, (b) the protecting group shouldbe stable under the reaction conditions selected for removing theα-amino protecting group at each step of the synthesis, and (c) the sidechain protecting group must be removable, upon the completion of thesynthesis containing the desired amino acid sequence, under reactionconditions that will not undesirably alter the peptide chain.

It should be possible to prepare many, or even all, of these peptidesusing recombinant DNA technology. However, when peptides are not soprepared, they are preferably prepared using the Merrifield solid-phasesynthesis, although other equivalent chemical syntheses known in the artcan also be used as previously mentioned. Solid-phase synthesis iscommenced from the C-terminus of the peptide by coupling a protectedα-amino acid to a suitable resin. Such a starting material can beprepared by attaching an α-amino-protected amino acid by an esterlinkage to a chloromethylated resin or a hydroxymethyl resin, or by anamide bond to a benzhydrylamine (BHA) resin orpara-methylbenzhydrylamine (MBHA) resin. Preparation of thehydroxymethyl resin is described by Bodansky et al. (1966).Chloromethylated resins are commercially available from Bio RadLaboratories (Richmond, Calif.) and from Lab. Systems, Inc. Thepreparation of such a resin is described by Stewart and Young (1969).BHA and MBHA resin supports are commercially available, and aregenerally used when the desired polypeptide being synthesized has anunsubstituted amide at the C-terminus. Thus, solid resin supports may beany of those known in the art, such as one having the formulae—O—CH₂-resin support, —NH BHA resin Support, or —NH-MBHA resin support.When the unsubstituted amide is desired, use of a BHA or MBHA resin ispreferred, because cleavage directly gives the amide. In case theN-methyl amide is desired, it can be generated from an N-methyl BHAresin. Should other substituted amides be desired, the teaching of U.S.Pat. No. 4,569,967 (Kornreich et al., 1986) can be used, or should stillother groups than the free acid be desired at the C-terminus, it may bepreferable to synthesize the peptide using classical methods as setforth in the Houben-Weyl text (1974).

The C-terminal amino acid, protected by Boc or Fmoc and by a side-chainprotecting group, if appropriate, can be first coupled to achloromethylated resin according to the procedure set forth in K. Horikiet al. (1978), using KF in DMF at about 60° C. for 24 hours withstirring, when a peptide having free acid at the C-terminus is to besynthesized. Following the coupling of the BOC-protected amino acid tothe resin support, the α-amino protecting group is removed, as by usingtrifluoroacetic acid (TFA) in methylene chloride or TFA alone. Thedeprotection is carried out at a temperature between about 0° C. androom temperature. Other standard cleaving reagents, such as HCI indioxane, and conditions for removal of specific α-amino protectinggroups may be used as described in Schroder & Lubke (1965).

After removal of the α-amino-protecting group, the remaining α-amino-and side chain-protected amino acids are coupled step-wise in thedesired order to obtain the intermediate compound defined hereinbefore,or as an alternative to adding each amino acid separately in thesynthesis, some of them may be coupled to one another prior to additionto the solid phase reactor. Selection of an appropriate coupling reagentis within the skill of the art. Particularly suitable as a couplingreagent is N,N′-dicyclohexylcarbodiimide (DCC, DIC, HBTU, HATU, TBTU inthe presence of HoBt or HoAt).

The activating reagents used in the solid phase synthesis of thepeptides are well known in the peptide art. Examples of suitableactivating reagents are carbodiimides, such asN,N′-diisopropylcarbodiimide andN-ethyl-N′-(3-dimethylaminopropyl)carbodiimide. Other activatingreagents and their use in peptide coupling are described by Schroder &Lubke (1965) and Kapoor (1970).

Each protected amino acid or amino acid sequence is introduced into thesolid-phase reactor in about a twofold or more excess, and the couplingmay be carried out in a medium of dimethylformamide (DMF):CH₂Cl₂ (1:1)or in DMF or CH₂Cl₂ alone. In cases where intermediate coupling occurs,the coupling procedure is repeated before removal of the α-aminoprotecting group prior to the coupling of the next amino acid. Thesuccess of the coupling reaction at each stage of the synthesis, ifperformed manually, is preferably monitored by the ninhydrin reaction,as described by Kaiser et al. (1970). Coupling reactions can beperformed automatically, as on a Beckman 990 automatic synthesizer,using a program such as that reported in Rivier et al. (1978).

After the desired amino acid sequence has been completed, theintermediate peptide can be removed from the resin support by treatmentwith a reagent, such as liquid hydrogen fluoride or TFA (if using Fmocchemistry), which not only cleaves the peptide from the resin but alsocleaves all remaining side chain protecting groups and also the α-aminoprotecting group at the N-terminus if it was not previously removed toobtain the peptide in the form of the free acid. If Met is present inthe sequence, the Boc protecting group is preferably first removed usingtrifluoroacetic acid (TFA)/ethanedithiol prior to cleaving the peptidefrom the resin with HF to eliminate potential S-alkylation. When usinghydrogen fluoride or TFA for cleaving, one or more scavengers such asanisole, cresol, dimethyl sulfide and methylethyl sulfide are includedin the reaction vessel.

Cyclization of the linear peptide is preferably affected, as opposed tocyclizing the peptide while a part of the peptido-resin, to create bondsbetween Cys residues. To effect such a disulfide cyclizing linkage,fully protected peptide can be cleaved from a hydroxymethylated resin ora chloromethylated resin support by ammonolysis, as is well known in theart, to yield the fully protected amide intermediate, which isthereafter suitably cyclized and deprotected. Alternatively,deprotection, as well as cleavage of the peptide from the above resinsor a benzhydrylamine (BHA) resin or a methylbenzhydrylamine (MBHA), cantake place at 0° C. with hydrofluoric acid (HF) or TFA, followed byoxidation as described above.

The peptides are also synthesized using an automatic synthesizer. Aminoacids are sequentially coupled to an MBHA Rink resin (typically 100 mgof resin) beginning at the C-terminus using an Advanced Chemtech 357Automatic Peptide Synthesizer. Couplings are carried out using1,3-diisopropylcarbodimide in N-methylpyrrolidinone (NMP) or by2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU) and diethylisopro-pylethylamine (DIEA). The FMOC protecting groupis removed by treatment with a 20% solution of piperidine indimethylformamide(DMF). Resins are subsequently washed with DMF (twice),followed by methanol and NMP.

Muteins, analogs or active fragments, of the foregoing conotoxinpeptides are also contemplated here. See, e.g., Hammerland et al, Eur.J. Pharmacol., 226, pp. 239-244 (1992). Derivative muteins, analogs oractive fragments of the conotoxin peptides may be synthesized accordingto known techniques, including conservative amino acid substitutions,such as outlined in U.S. Pat. No. 5,545,723 (see particularly col. 2,line 50—col. 3, line 8); U.S. Pat. No. 5,534,615 (see particularly col.19, line 45—col. 22, line 33); and U.S. Pat. No. 5,364,769 (seeparticularly col. 4, line 55—col. 7, line 26), each herein incorporatedby reference.

Pharmaceutical compositions containing a compound of the presentinvention or its pharmaceutically acceptable salts as the activeingredient can be prepared according to conventional pharmaceuticalcompounding techniques. See, for example, Remington's PharmaceuticalSciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa.). Typically,an antagonistic amount of the active ingredient will be admixed with apharmaceutically acceptable carrier. The carrier may take a wide varietyof forms depending on the from of preparation desired foradministration, e.g., intravenous, oral or parenteral. The compositionsmay further contain antioxidizing agents, stabilizing agents,preservatives and the like. For examples of delivery methods see U.S.Pat. No. 5,844,077, incorporated herein by reference.

For oral administration, the compounds can be formulated into solid orliquid preparations such as capsules, pills, tablets, lozenges, melts,powders, suspensions or emulsions. In preparing the compositions in oraldosage form, any of the usual pharmaceutical media may be employed, suchas, for example, water, glycols, oils, alcohols, flavoring agents,preservatives, coloring agents, suspending agents, and the like in thecase of oral liquid preparations (such as, for example, suspensions,elixirs and solutions); or carriers Such as starches, sugars, diluents,granulating agents, lubricants, binders, disintegrating agents and thelike in the case of oral solid preparations (such as, for example,powders, capsules and tablets). Because of their ease in administration,tablets and capsules represent the most advantageous oral dosage unitform, in which case solid pharmaceutical carriers are obviouslyemployed. If desired, tablets may be sugar-coated or enteric-coated bystandard techniques. The active agent can be encapsulated to make itstable to passage through the gastrointestinal tract while at the sametime allowing for passage across the blood brain barrier. See forexample, WO 96/11698.

For parenteral administration, the compound may be dissolved in apharmaceutical carrier and administered as either a solution or asuspension. Illustrative of suitable carriers are water, saline,dextrose solutions, fructose solutions, ethanol, or oils of animal,vegetative or synthetic origin. The carrier may also contain otheringredients, for example, preservatives, suspending agents, solubilizingagents, buffers and the like. When the compounds are being administeredintrathecally, they may also be dissolved in cerebrospinal fluid.

The active agent is preferably administered in an therapeuticallyeffective amount. The actual amount administered, and the rate andtime-course of administration, will depend on the nature and severity ofthe condition being treated. Prescription of treatment, e.g. decisionson dosage, timing, etc., is within the responsibility of generalpractitioners or spealists, and typically takes account of the disorderto be treated, the condition of the individual patient, the site ofdelivery, the method of administration and other factors known topractitioners. Examples of techniques and protocols can be foundRemington's Pharmaceutical Sciences. Typically the active agents of thepresent invention exhibit their effect at a dosage range from about0.001 mg/kg to about 250 mg/kg, preferably from about 0.01 mg/kg toabout 100 mg/kg of the active ingredient, more preferably from a bout0.05 mg/kg to about 75 mg/kg. A suitable dose can be administered inmultiple sub-doses per day. Typically, a dose or sub-dose may containfrom about 0.1 mg to about 500 mg of the active ingredient per unitdosage form. A more preferred dosage will contain from about 0.5 mg toabout 100 mg of active ingredient per unit dosage form. Dosages aregenerally initiated at lower levels and increased until desired effectsare achieved.

Alternatively, targeting therapies may be used to deliver the activeagent more specifically to certain types of cell, by the use oftargeting systems such as antibodies or cell specific ligands. Targetingmay be desirable for a variety of reasons, e.g. if the agent isunacceptably toxic, or if it would otherwise require too high a dosage,or if it would not otherwise be able to enter the target cells.

The active agents, which are peptides, can also be administered in acell based delivery system in which a DNA sequence encoding an activeagent is introduced into cells designed for implantation in the body ofthe patient, especially in the spinal cord region. Suitable deliverysystems are described in U.S. Pat. No. 5,550,050 and published PCTApplication Nos. WO 92/19195, WO 94/25503, WO 95/01203, WO 95/05452, WO96/02286, WO 96/02646, WO 96/40871, WO 96/40959 and WO 97/12635.Suitable DNA sequences can be prepared synthetically for each activeagent on the basis of the developed sequences and the known geneticcode.

EXAMPLES

The present invention is described by reference to the followingExamples, which are offered by way of illustration and are not intendedto limit the invention in any manner. Standard techniques well known inthe art or the techniques specifically described below were utilized.

Example 1 Isolation of τ-Conotoxins

Crude venom was extracted from venom ducts (Cruz et al., 1976), and thecomponents were purified as previously described (Cartier et al., 1996).The crude extract from venom ducts was purified by reverse phase liquidchromatography (RPLC) using a Vydac C₁₈ semi-preparative column (10×250mm). Further purification of bioactive peaks was done on a Vydac C₁₈analytical column (4.6×220 mm). The effluents were monitored at 220 nm.Peaks were collected, and aliquots were assayed for activity.

The amino acid sequence of the purified peptides were determined bystandard methods. The purified peptides were reduced and alkylated priorto sequencing by automated Edman degradation on an Applied Biosystems477A Protein Sequencer with a 120A Analyzer (DNA/Peptide Facility,University of Utah) (Martinez et al., 1995; Shon et al., 1994).

In accordance with this method, peptides AuVA, AuVB and PVA wereobtained.

Example 2 Synthesis of Conopeptides

The synthesis of conopeptides, either the mature toxins or the precursorpeptides, was separately performed using conventional protectionchemistry as described by Cartier et al. (1996). Briefly, the linearchains were built on Rink amide resin by Fmoc procedures with2-(1H-benzotriol-1-yl)-1,1,3,3,-tetramethyluronium tetrafluoroboratedcoupling using an ABI model 430A peptide sythesizer with amino acidderivatives purchased from Bachem (Torrence Calif.). Orthogonalprotection was used on cysteines: two cysteines were protected as thestable Cys(S-acetamidomethyl), while the other two cysteines wereprotected as the acid-labile Cys(S-trityl). After removal of theterminal Fmoc protecting group and cleavage of the peptides from theresins, the released peptides were precipitated by filtering thereaction mixture into −10° C. methyl t-butyl ether, which removed theprotecting groups except the Cys(S-acetamidomethyl). The peptides weredissolved in 0.1% TFA and 60% acetonitrile and purified by RPLC on aVydac C₁₈ preparative column (22×250 mm) and eluted at a flow rate of 20mL/min with a gradient of acetonitrile in 0.1% TFA.

The disulfide bridges in the three conopeptides were formed as describedin Cartier et al. (1996). Briefly, the disulfide bridges between onepair of cysteines were formed by air oxidation which was judged to becomplete by analytical RPLC. The monocyclic peptides were purified byRPLC on a Vydac C₁₈ prepartive column (22×250 mm) and eluted with agradient of acetonitrile in 0.1% TFA. Removal of S-acetamidomethylgroups and closure of the disulfide bridge between the other pair ofcysteines was carried out simultaneously be iodine oxidation. The cyclicpeptides were purified by RPLC on a Vydac C₁₈ prepartive column (22×250mm) and eluted with a gradient of acetonitrile in 0.1% TFA.

Example 3 Isolation of DNA Encoding τ-Conotoxins

DNA coding for τ-conotoxins was isolated and cloned in accordance withconventional techniques using general procedures well known in the art,such as described in Olivera et al. (1996). Alternatively, cDNAlibraries was prepared from Conus venom duct using conventionaltechniques. DNA from single clones was amplified by conventionaltechniques using primers which correspond approximately to the M13universal priming site and the M13 reverse universal priming site.Clones having a size of approximately 300-500 nucleotides were sequencedand screened for similarity in sequence to known τ-conotoxins isolatedin Example 1. The DNA sequences and encoded propeptide sequences are setforth in Tables 1-15. DNA sequences coding for the mature toxin can alsobe prepared on the basis of the DNA sequences set forth in these Tables.

TABLE 1 DNA Sequence (SEQ ID NO:20) and Protein Sequence (SEQ ID NO:21)of Tx5.1 ggtactcaac gaacttcaag acacattctt ttcacctgga cacgggaagctgactacaag caga atg tgc tgt ctc cca gtg ttc gtc att ctt ctg ctg ctg attgca      Met Cys Cys Leu Pro Val Phe Val Ile Leu Leu Leu Leu Ile Ala tctgca cct agc gtt gat gcc caa ccg aag acc aaa gat gat gtg ccc Ser Ala ProSer Val Asp Ala Gln Pro Lys Thr Lys Asp Asp Val Pro ctg gca cct ttg cacgat aat gca aag agt gca cta caa cat ttg aac Leu Ala Pro Leu His Asp AsnAla Lys Ser Ala Leu Gln His Leu Asn caa cgc tgc tgc caa aca ttc tat tggtgc tgt gtt caa ggg aaa Gln Arg Cys Cys Gln Thr Phe Tyr Trp Cys Cys ValGln Gly Lys tgaatttgga tgagacccct gcgaactgtc catggatgtg agatttggaaagcagactgt tcctttcgca cgtgttcgtg gaattttgaa tggtcgttaa caacacgctgccacttgcaa gctactatct ctctgtcctt tcatctgtgg aactggatga cctaacaactgaaatatcat agaaattttt cagtgggtat acactatgac catgtagtca gtaattacatcatttggacc ttttgaaata tttttcaaaa tgttaagatt tttcccccng gaaaggncttttgaagtaaa tatt

TABLE 2 DNA Sequence (SEQ ID NO:22) and Protein Sequence (SEQ ID NO:23)of G5.1 atg tgc tgt ctc cca gtc ttc gtc att ctt ctg ttg ctg att aca tctMet Cys Cys Leu Pro Val Phe Val Ile Leu Leu Leu Leu Ile Thr Ser gca cctagc gtt gat gct cta ccg aag acc agg gat gat gtg ccc cta Ala Pro Ser ValAsp Ala Leu Pro Lys Thr Arg Asp Asp Val Pro Leu gca tct ttc cac ggt ggatat aat gca agg aga atc cta caa agg cgt Ala Ser Phe His Gly Gly Tyr AsnAla Arg Arg Ile Leu Gln Arg Arg cag ggc tgg tgc tgc aaa gaa aat att gcgtgc tgt ata tagtggtaac Gln Gly Trp Cys Cys Lys Glu Asn Ile Ala Cys CysIle gggaaatgac tttggatgag acccctgcaa actgtccctg gatgtgaaat ttggaaagtagactgttcct ttcgcgcgtg ttcgtggaat ttcaaatggt cgtcaacaac acactgctacttgcaaagct actatctctc tgtcctttca tctgtggaac tgggtgatct aacagctgaaatgtcgcaga aatttttcaa ttggtctata ctatgaccat gta

TABLE 3 DNA Sequence (SEQ ID NO:24) and Protein Sequence (SEQ ID NO:25)of Qc5.1 atg cgc tgt gtc cca gtc ttc atc att ctt ctg ctg ctg agt cca tctMet Arg Cys Val Pro Val Phe Ile Ile Leu Leu Leu Leu Ser Pro Ser gca cctagc gtt gat gcc cat ccg atg acc aaa gat gat gtg ccc cag Ala Pro Ser ValAsp Ala His Pro Met Thr Lys Asp Asp Val Pro Gln gca tca ttc cat gat gatgca aag cga acc cta caa gta cct tgg atg Ala Ser Phe His Asp Asp Ala LysArg Thr Leu Gln Val Pro Trp Met aaa cgc ggg tgc tgc gca agg ttg act tgctgc gtt gga cga Lys Arg Gly Cys Cys Ala Arg Leu Thr Cys Cys Val Gly Argtaaagggaaa tgactttgga tgagacccct gcgaactgtc cctggatgtg aaatttggacagcagaccgc tcctttcgca cgtgttcgtg gaattttgaa tggtcgttaa caacacgctgccacttgcaa gctattatct ctctgtccct ttatctgtgg aactggataa tctaacaactgaaatgtcat tgaaaatttt caatggatat atattatgat ccatata

TABLE 4 DNA Sequence (SEQ ID NO:26) and Protein Sequence (SEQ ID NO:27)of Im5.1 aattcggaag ctgactacaa gcaga atg tac tgt ctc cca gtc ttc atc att                            Met Tyr Cys Leu Pro Val Phe Ile Ile ctt ctgctg ctg att tca tct gca cct agc act cct ccc caa cca agg Leu Leu Leu LeuIle Ser Ser Ala Pro Ser Thr Pro Pro Gln Pro Arg aac aaa gat cgt gtg cacctg ata tct tta ctc gat aat cac aag caa Asn Lys Asp Arg Val His Leu IleSer Leu Leu Asp Asn His Lys Gln atc cta caa aga gat tgg aac agt tgc tgtggg aaa aat cct ggt tgc Ile Leu Gln Arg Asp Trp Asn Ser Cys Cys Gly LysAsn Pro Gly Cys tgt cct tgg gga aaa tgactttgga tgagacccct gcaaactgtccctggatgtg Cys Pro Trp Gly Lys agatttggaa agcagaccgt ttgtggaattttgaatggtc gttaacaaca cgctgccact tgcaagctac aatctctctg tcctttcatctttggaactg gatgatcaaa caactgaaat gtcatagaaa tttttcaatg ggtatacaatatgtgggcat ttagtcagta attacatcat ttgg

TABLE 5 DNA Sequence (SEQ ID NO:28) and Protein Sequence (SEQ ID NO:29)of G5.2 atg tgc tgt ctc cca gtc ttc gtc att ctt ctg ttg ctg att aca tctMet Cys Cys Leu Pro Val Phe Val Ile Leu Leu Leu Leu Ile Thr Ser gca cctagc gtt gat gct cta ccg aag acc agg gat gat gtg ccc cta Ala Pro Ser ValAsp Ala Leu Pro Lys Thr Arg Asp Asp Val Pro Leu gca tct ttc cac ggt ggatat aat gca agg aga atc cta caa agg cgt Ala Ser Phe His Gly Gly Tyr AsnAla Arg Arg Ile Leu Gln Arg Arg cag ggc tgg tgc tgc aaa gaa aat att gcgtgc tgt gta tagtggtaac Gln Gly Trp Cys Cys Lys Glu Asn Ile Ala Cys CysVal gggaaatgac tttggatgag acccctgcaa actgtccctg gatgtgaaat ttggaaagtagactgttcct ttcgcgcgtg ttcgtggaat ttcaaatggt cgtcaacaac acactgctacttgcaaagct actatctctc tgtcctttca tctgtggaac tgggtgatct aacagctgaaatgtcgcaga aatttttcaa ttggtctata ctatgaccat gtagtcag

TABLE 6 DNA Sequence (SEQ ID NO:30) and Protein Sequence (SEQ ID NO:31)of Tx5.2a atg cgc tgt ttc cca gtc ttc atc att ctt ctg ctg cta att gcatct Met Arg Cys Phe Pro Val Phe Ile Ile Leu Leu Leu Leu Ile Ala Ser gcacct tgc ttt gat gcc cga acg aag acc gat gat gat gtg ccc ctg Ala Pro CysPhe Asp Ala Arg Thr Lys Thr Asp Asp Asp Val Pro Leu tca tct ctc cgc gataat cta aag cga acg ata cga aca cgc ctg aac Ser Ser Leu Arg Asp Asn LeuLys Arg Thr Ile Arg Thr Arg Leu Asn ata cgc gag tgc tgc gag gat gga tggtgc tgt act gct gca ccc tta Ile Arg Glu Cys Cys Glu Asp Gly Trp Cys CysThr Ala Ala Pro Leu aca ggt cgt tagggataaa ggaaaatggc tttggatgagacccctgcga Thr Gly Arg attgtccctg gatgtgagat ttggaaagca gactgttcctttcgcacgtg ttcgtggaat ttcgaatggt cgttaacaac acgctgccac ttgcaagccaccatctctct gtcctttcgt atgtggaact gtatgatcta acaactgaaa tgtcagaaagttttcagtgg gtatacacta tgatcgtata

TABLE 7 DNA Sequence (SEQ ID NO:32) and Protein Sequence (SEQ ID NO:33)of Tx5.2b atg cgc tgt ttc cca gtc ttc atc att ctt ctg ttg cta att gcatct Met Arg Cys Phe Pro Val Phe Ile Ile Leu Leu Leu Leu Ile Ala Ser gcacct tgc ttt gat gcc cga acg aag acc gat gat gat gtg ccc ctg Ala Pro CysPhe Asp Ala Arg Thr Lys Thr Asp Asp Asp Val Pro Leu tca tct ctc cgc gataat cta aag cga acg ata cga aca cgc ctg aac Ser Ser Leu Arg Asp Asn LeuLys Arg Thr Ile Arg Thr Arg Leu Asn ata cgc ggg tgc tgc gag gat gga tggtgc tgt act gct gca ccc tta Ile Arg Gly Cys Cys Glu Asp Gly Trp Cys CysThr Ala Ala Pro Leu aca ggt cgt tagggataaa ggaaaatggc tttggatgagacccctgcaa Thr Gly Arg attgtccctg gatgtgagat ttggaaagca gactgttcctttcgcacgtg ttcgtggaat ttcgaatggt cgttaacaac acgctgccac ttgcaagccaccatctctct gtcctttcgt atgtggaact gtatgatcta acaactgaaa tgtcagaaagttttcagtgg gtatacacta tgatcgtata gtcagtaatt

TABLE 8 DNA Sequence (SEQ ID NO:34) and Protein Sequence (SEQ ID NO:35)of Mr5.1 atg cgc tgc ctc cca gtc ttc gtc att ctt ctg ctg ctg att gca tctMet Arg Cys Leu Pro Val Phe Val Ile Leu Leu Leu Leu Ile Ala Ser gca cctagc gtt gat gcc cga ccg aag acc aaa gat gat atg ccc ctg Ala Pro Ser ValAsp Ala Arg Pro Lys Thr Lys Asp Asp Met Pro Leu gca tct ttc cat gat aatgca aag cga atc ctg caa ata ctt cag gac Ala Ser Phe His Asp Asn Ala LysArg Ile Leu Gln Ile Leu Gln Asp aga aat ggt tgc tgc aga gca gga gac tgctgt tca cga ttt gag ata Arg Asn Gly Cys Cys Arg Ala Gly Asp Cys Cys SerArg Phe Glu Ile aag gaa aat gac ttt gga tgagacccct gcaaactgtc cttggatgtgLys Glu Asn Asp Phe Gly agatttggaa agcagactgt tcctttcgca cgtgttcgtggaatttcgaa tggtcgttaa caacacgctg ccacttgcaa gctactatct ctctgtccttttgtctgtgg aactgtatga tcaaacaact gaaatgtcat agaaattttt cagtgggtaaacactatgac catgta

TABLE 9 DNA Sequence (SEQ ID NO:36) and Protein Sequence (SEQ ID NO:37)of Mr5.2 ga atg cgc tgc ctc cca gtc ttc gtc att ctt ctg ctg ctg att gca   Met Arg Cys Leu Pro Val Phe Val Ile Leu Leu Leu Leu Ile Ala tct gcacct agc gtt gat gcc cga ccg aag acc aaa gat gat atg ccc Ser Ala Pro SerVal Asp Ala Arg Pro Lys Thr Lys Asp Asp Met Pro ctg gca tct ttc cac gataat gca aag cga atc ctg caa ata ctt cag Leu Ala Ser Phe His Asp Asn AlaLys Arg Ile Leu Gln Ile Leu Gln gac aga aat gct tgc tgc ata gta agg cagtgc tgt tgatgatttg Asp Arg Asn Ala Cys Cys Ile Val Arg Gln Cys Cysagataaagga aaatgacttt ggatgagacc cctgcaaact gtccctggat gtgagatttggaaagcagac tgttcctttc gcacgtgttc gtggaatttc gaatggtcgt taacaacacgctgccacttg caagctacta tctctctgtc ctttcatctg tggaactgta tgatcaaacaactgaaatgt catagaaatt tttcagtggg taaacactat gatcatgtag tcagtaattacatcatttgg aattccatca agcttatcga taccgtcgac ctcgaggggg ggcccggt

TABLE 10 DNA Sequence (SEQ ID NO:38) and Protein Sequence (SEQ ID NO:39)of Mr5.3 atg cgc tgc ctc cca gtc ttt gtc att ctt ctg ctg ctg att gca tctMet Arg Cys Leu Pro Val Phe Val Ile Leu Leu Leu Leu Ile Ala Ser gca cctagc gtt gat gcc cga ccg aag acc aaa gat gat atg ccc ctg Ala Pro Ser ValAsp Ala Arg Pro Lys Thr Lys Asp Asp Met Pro Leu gca tct ttc cat gat aatgca aag cga atc ctg caa ata ctt cag gac Ala Ser Phe His Asp Asn Ala LysArg Ile Leu Gln Ile Leu Gln Asp aga aat ggt tgc tgc aga gca gga gac tgctgt tca tgatttgaga Arg Asn Gly Cys Cys Arg Ala Gly Asp Cys Cys Sertaaagggaaa tgactttgga tgagacccct gcaaactgtc cttggatgtg agatttggaaagcagactgt tcctttcgca cgtgttcgtg gaatttcgaa tggtcgttaa caacacgctgccacttgcaa gctactatct ctctgtcctt tcatctgtgg aactgtatga tcaaacaact

TABLE 11 DNA Sequence (SEQ ID NO:40) and Protein Sequence (SEQ ID NO:41)of Ca5.1 atg cgc tgt ctc ccg gtc ttc atc att ctt ctg ctg ctg att gca tctMet Arg Cys Leu Pro Val Phe Ile Ile Leu Leu Leu Leu Ile Ala Ser gca cctggc gtt gat gcc caa ccg aag acc aaa tat aat gcg ccc ctg Ala Pro Gly ValAsp Ala Gln Pro Lys Thr Lys Tyr Asn Ala Pro Leu aca tct ctc cac gat aatgca aag ggt ata cta caa gaa cat tgg aac Thr Ser Leu His Asp Asn Ala LysGly Ile Leu Gln Glu His Trp Asn aaa cgc tgc tgc ccc aga agg ctt gcc tgctgt att ata gga cgg aaa Lys Arg Cys Cys Pro Arg Arg Leu Ala Cys Cys IleIle Gly Arg Lys tgaatgattt tgggtgagat ccctgcaaac tgtccctgga tttgaattttggaaagcaga ctgttccttt cgcacgtgtt cgtggaattt cgaatggtcg ttaacaacacgctgccactt gcaagctact atctctctgt cctttttctc tgtgaaactg gatggtctaacaactgaaat gtcatagaaa attttcaatg ggtatactct atgaccatct a

TABLE 12 DNA Sequence (SEQ ID NO:42) and Protein Sequence (SEQ ID NO:43)of Ca5.2 atg cgc tgt ctc cca gtc ttc atc att ctt ctg ctg ctg att gca tctMet Arg Cys Leu Pro Val Phe Ile Ile Leu Leu Leu Leu Ile Ala Ser gca cctggc gtt gat gcc caa ccg aag acc aaa tat gat gcg ccc ctg Ala Pro Gly ValAsp Ala Gln Pro Lys Thr Lys Tyr Asp Ala Pro Leu aca tct ctc cac gat aatgca aag ggt ata cta caa gaa cat tgg aac Thr Ser Leu His Asp Asn Ala LysGly Ile Leu Gln Glu His Trp Asn aaa cgc tgc tgc ccc aac aag cct tgc tgtttt ata gga agg aaa Lys Arg Cys Cys Pro Asn Lys Pro Cys Cys Phe Ile GlyArg Lys tgaatgattt tgggtgagac ccctgcaaac tgtccctgga tttgaattttggaaagcaga ctgttccttt cgcacgtgtt cgtggaattt cgaatggtcg ttaacaacacgctgccactt gcaagctact atctctctgt cctttttctc tgtgaaactg gatggtctaacaactgagat gtcatagaaa attttcaatc ggtgtactct atgaccatct a

TABLE 13 DNA Sequence (SEQ ID NO:44) and Protein Sequence (SEQ ID NO:45)of Qc5.2 atg cgc tgt gtc cca gtc ttc atc att ctt ctg ctg ctg agt cca tctMet Arg Cys Val Pro Val Phe Ile Ile Leu Leu Leu Leu Ser Pro Ser gca cctagc gtt gat gcc cat ccg atg acc aaa gat gat gta ccc cag Ala Pro Ser ValAsp Ala His Pro Met Thr Lys Asp Asp Val Pro Gln gca tct ctc cat gat gatgca aag cga acc cta caa gta cct tgg atg Ala Ser Leu His Asp Asp Ala LysArg Thr Leu Gln Val Pro Trp Met aaa cgc ggg tgc tgc gca atg ttg act tgctgc gtt gga cga Lys Arg Gly Cys Cys Ala Met Leu Thr Cys Cys Val Gly Argtaaagggaaa tgactttgga tgagacccct acgaactgtc cctggatgtg aaatttggacagcagactgc tcctttcgca cgtgttcgtg gaatttcgaa tggtcgttaa caacacgctgccacttgcaa gctattatct ctctgtccct ttatctgtgg aactggataa tctaacaactgaaacgtcat tgaaaatttt caatggatat atattatgat ccatata

TABLE 14 DNA Sequence (SEQ ID NO:46) and Protein Sequence (SEQ ID NO:47)of Gm5.1 gggcaggtac tcaacgaact tcaggacaca ttcttttcac ctggacacgggaaactgact ataagcaga atg cgc tac cta cca gtc ttc gtc att ctt ctg ctg ctgatt           Met Arg Tyr Leu Pro Val Phe Val Ile Leu Leu Leu Leu Ilegca tct ata cct agc gat act gtc caa ctg aag acc aaa gat gat atg Ala SerIle Pro Ser Asp Thr Val Gln Leu Lys Thr Lys Asp Asp Met ccc ctg gca tctttc cac ggt aat gga aga cga atc ctg cga atg ctt Pro Leu Ala Ser Phe HisGly Asn Gly Arg Arg Ile Leu Arg Met Leu tca aac aaa cgc tta tgc tgt gtcacc gag gat tgg tgc tgt gaa tgg Ser Asn Lys Arg Leu Cys Cys Val Thr GluAsp Trp Cys Cys Glu Trp tgg taaaggaaaa tgactttgga tgagacccct gcaaactgtttctggatgtg Trp agatttggaa agcagactgt tctttcgcac gtattcgtga aatttcgaatggtcgttaac aacacgctgc cacttgcaag ctgctatctc tctgtctttt catctgtggaactgtatgat ctaacaactg aaatgtcata gacatttttc attgggtata cactatgaccatgtagccag taattacatc atttggacct tttggatatt tttcagtatg taagtgtgttcccttaaaaa gtcctttgta attatgtatt ttaanaattt angttttgca cataaattgtaaaacgctgt cctttctgtt gntcctacat cantggtggg gaaaagnaaa atgtttggccntggtcaaat ttaaataatn accctgccgt ttnaatgcng ttattantgg tattttnaacnttgnacggt taaactt

TABLE 15 DNA Sequence (SEQ ID NO:48) and Protein Sequence (SEQ ID NO:49)of Gm5.2 ga atg cgc tgt ctc cca gtc ttc gtc att ctt ctg ctg ctg att gca   Met Arg Cys Leu Pro Val Phe Val Ile Leu Leu Leu Leu Ile Ala tct gcacct agc gtt gat gcc caa ccg aag acc aaa gat gat gtg ccc Ser Ala Pro SerVal Asp Ala Gln Pro Lys Thr Lys Asp Asp Val Pro ctg gca cct ttg cac gataat ata agg agt act cta caa aca ctt cgg Leu Ala Pro Leu His Asp Asn IleArg Ser Thr Leu Gln Thr Leu Arg aag aaa gtc tgc tgc cgc cca gtg cag gattgc tgt tca ggg aaa Lys Lys Val Cys Cys Arg Pro Val Gln Asp Cys Cys SerGly Lys tgaagggaaa tgaatttgga tgagacccct gcgaactgtc cctggatgtgagatttggaa agcagactgt tcctttcgca cgtgttcgtg gaatttcgaa tggtcgttaacaacacgctg ccacttgcaa gctactatct ctctgtcctt tcatctgcgg aactggatgacctaaagctt gtgatc

Example 4 Biological Activity of τ-Conotoxins

The biological activity of τ-conotoxin peptides at the acetylcholinereceptor was tested in the fluorescence assay as described byCornell-Bell et al. (1999). Briefly, primary cortical cells are exposedto acetylcholine in the presence or absence of a τ-conotoxin peptide.Acetylcholine causes the primary cortical cells to flux calcium, whichis measured by increases in fluorescence in cells loaded with Fluo-3, acalcium imaging dye. The τ-conotoxin peptide AuVA inhibited the responseof primary cortical cells to acetylcholine at low concentrations (10 pM)at 15 seconds following exposure to the peptide and acetylcholine. Thisstudy shows that the τ-conotoxin peptide act at the acetylcholinereceptor.

Example 5 Effect of τ-Conotoxins in a Pain Model

The effect of τ-conotoxin peptides for use in treating pain was bytesting in two pain models, the first being the hind-paw licking model(Woolfe and MacDonald, 1944; Plummer et al., 1991; Suh et al., 1992;Plone et al., 1996) and the second being the accelerating roto-rodmodel. In the hind-paw licking model, it was found that 10 nmol ofτ-conotoxin peptide AuVA increased the latency to initiate hind-pawlicking in mice on a 55° C. hot plate 15 minutes following freehandi.c.v. injection. It was further found that 1 nmol τ-conotoxin peptideAuVA did not have any effect in this model. In the accelerating roto-rodmodel, it was found that τ-conotoxin peptide AuVA produced impairment ofmotor performance following injection of τ-conotoxin peptide AuVA. Theeffects seen in these models demonstrates that the τ-conotoxin peptideshave analgesic properties.

It will be appreciated that the methods and compositions of the instantinvention can be incorporated in the form of a variety of embodiments,only a few of which are disclosed herein. It will be apparent to theartisan that other embodiments exist and do not depart from the spiritof the invention. Thus, the described embodiments are illustrative andshould not be construed as restrictive.

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1. A method for inducing analgesia in an individual which comprisesadministering an effective amount of a substantially pure τ-conotoxinpeptide having the generic formula I:Xaa₁-Xaa₂-Xaa₃-Xaa₄-Cys-Cys-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Cys-Cys-Xaa₁₀-Xaa₁₁-Xaa₁₂-Xaa₁₃-Xaa₁₄-Xaa₁₅-Xaa₁₆-Xaa₁₇-Xaa₁₈-Xaa₁₉(SEQ ID NO:1), wherein Xaa₁ is des-Xaa₁, Asp, Glu or γ-carboxy-Glu(Gla); Xaa₂ is des-Xaa₂, Gln, Asn, Glu, Trp (D or L), neo-Trp, halo-Trpor any unnatural aromatic amino acid; Xaa₃ is des-Xaa₃, Gly, Ala, Asn orGln; Xaa₄ is des-Xaa₄, Val, Leu (D or L), Ile, Ala, Gly, Glu, Gla, Asp,Ser, Thr, Phe, Trp (D or L), neo-Trp, halo-Trp (D or L) or any unnaturalaromatic amino acid; Xaa₅ is Pro, hydroxy-Pro, Gln, Asn, Glu, Gla, Ala,Gly, Lys, Arg, Ile, Val, homoarginine, ornithine, N-methyl-Lys,N,N-dimethyl-Lys, N,N,N-trimethyl-Lys or any unnatural basic amino acid;Xaa₆ is Val, Phe, Thr, Ser, Glu, Gla, Asp, Asn, Gln, Ala, Gly, Ile, Leu(D or L), Met, Pro, hydroxy-Pro, Arg, homoarginine, ornithine, Lys,N-methyl-Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys, any unnatural basicamino acid or any unnatural aromatic amino acid; Xaa₇ is any Val, Ile,Asn, Leu (D or L), Gln, Gly, Ala, Phe, Glu, Gla, Arg, ornithine,homoarginine, Lys, N-methy-Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys,any unnatural basic amino acid or any unnatural aromatic amino acid;Xaa₈ is Ile, Leu (D or L), Met, Thr, Ser, Pro, hydroxy-Pro, Gln, Asp,Glu, Gla, Asn, Arg, homoarginine, ornithine, Lys, N-methyl-Lys,N,N-dimethyl-Lys, N,N,N-trimethyl-Lys, Tyr, nor-Tyr, mono-halo-Tyr,di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr, any unnatural basicamino acid, any unnatural aromatic amino acid or any unnatural hydroxycontaining amino acid; Xaa₉ is des-Xaa₉, Ala, Gly, Asp, Glu, Gla, Trp (Dor L) neo-Trp, halo-Trp (D or L), Lys, N-methy-Lys, N,N-dimethyl-Lys,N,N,N-trimethyl-Lys, Arg, homoarginine, ornithine, Tyr, nor-Tyr,mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr orany unnatural basic amino acid; Xaa₁₀ is des-Xaa₁₀, Ile, Leu (D or L),Val, Glu, Gla, Asp, Thr, Ser, Pro, hydroxy-Pro, Trp (D or L), neo-Trp,halo-Trp (D or L), Phe, any unnatural aromatic amino acid or anyunnatural hydroxy containing amino acid; Xaa₁₁ is des-Xaa₁₁, Gln, Asn,Leu (D or L), Ile, Val, Ala, Gly, Trp (D or L), neo-Trp, halo-Trp (D orL), Arg, homoarginine, ornithine, Lys, N-methy-Lys, N,N-dimethyl-Lys,N,N,N-trimethyl-Lys, any unnatural basic amino acid or any unnaturalaromatic amino acid; Xaa₁₂ is des-Xaa₁₂, Ala, Gly, Phe, Trp (D or L),neo-Trp, halo-Trp (D or L) or any unnatural aromatic amino acid; Xaa₁₃is des-Xaa₁₃, Glu, Gla, Asp, Phe or any unnatural aromatic amino acid;Xaa₁₄ is des-Xaa₁₄, Ile, Val or Leu (D or L); Xaa₁₅ is des-Xaa₁₅, Thr,Ser, Arg, homoarginine, ornithine, Lys, N-methy-Lys, N,N-dimethyl-Lys,N,N,N-trimethyl-Lys or any unnatural basic amino acid; Xaa₁₆ isdes-Xaa₁₆, Glu, Gla or Asp; Xaa₁₇ is des-Xaa₁₇, Asn or Gln; Xaa₁₈ isdes-Xaa₁₈, Asp, Glu or Gla; Xaa₁₉ is des-Xaa₁₉, Phe or any unnaturalaromatic amino acid; and the C-terminus contains a free carboxyl groupor an amide group.
 2. The method of claim 1, wherein the peptide isselected for the group consisting of: (SEQ ID NO:2);Phe-Cys-Cys-Xaa₁-Val-Ile-Arg-Xaa₂-Cys-Cys-Xaa₃ (SEQ ID NO:3);Phe-Cys-Cys-Xaa₁-Phe-Ile-Arg-Xaa₂-Cys-Cys-Xaa₃ (SEQ ID NO:4);Cys-Cys-Gln-Thr-Phe-Xaa₂-Xaa₃-Cys-Cys-Gln (SEQ ID NO:5);Xaa₄-Gly-Xaa₃-Cys-Cys-Xaa₅-Xaa₆-Asn-Ile-Ala-Cys- Cys-Ile (SEQ ID NO:6);Gly-Cys-Cys-Ala-Arg-Leu-Thr-Cys-Cys-Val (SEQ ID NO:7);Asn-Gly-Cys-Cys-Xaa₁-Xaa₅-Gln-Met-Arg-Cys-Cys-Thr (SEQ ID NO:8);Asp-Xaa₃-Asn-Ser-Cys-Cys-Gly-Xaa₅-Asn-Xaa₁-Gly- Cys-Cys-Xaa₁-Xaa₃ (SEQID NO:9); Xaa₄-Gly-Xaa₃-Cys-Cys-Xaa₅-Xaa₆-Asn-Ile-Arg- Cys-Cys-Val (SEQID NO:10); Xaa₆-Cys-Cys-Xaa₆-Asp-Gly-Xaa₃-Cys-Cys-Thr-Ala-Ala-Xaa₁-Leu-Thr (SEQ ID NO:11);Gly-Cys-Cys-Xaa₆-Asp-Gly-Xaa₃-Cys-Cys-Thr-Ala-Ala-Xaa₁-Leu-Thr (SEQ IDNO:12); Asn-Gly-Cys-Cys-Arg-Ala-Gly-Asp-Cys-Cys-Ser-Arg-Phe-Xaa₆-Ile-Xaa₅-Xaa₆-Asn-Asp-Phe (SEQ ID NO:13);Asn-Ala-Cys-Cys-Ile-Val-Arg-Gln-Cys-Cys (SEQ ID NO:14);Asn-Gly-Cys-Cys-Arg-Ala-Gly-Asp-Cys-Cys-Ser (SEQ ID NO:15);Cys-Cys-Xaa₁-Arg-Arg-Leu-Ala-Cys-Cys-Ile-Ile (SEQ ID NO:16);Cys-Cys-Xaa₁-Asn-Xaa₅-Xaa₁-Cys-Cys-Phe-Ile (SEQ ID NO:17);Gly-Cys-Cys-Ala-Met-Leu-Thr-Cys-Cys-Val (SEQ ID NO:18); andLeu-Cys-Cys-Val-Thr-Xaa₆-Asp-Xaa₃-Cys-Cys-Xaa₆- Xaa₃-Xaa₃ (SEQ IDNO:19); Val-Cys-Cys-Arg-Xaa₁-Val-Gln-Asp-Cys-Cys-Ser.

wherein Xaa₁ is Pro or hydroxy-Pro; Xaa₂ is Tyr, mono-halo-Tyr,di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr or nitro-Tyr; Xaa₃ is Trp orhalo-Trp; Xaa₄ is Gln or pyro-Glu; Xaa₅ is Lys, N-methyl-Lys,N,N-dimethyl-Lys or N,n,N-trimethyl-Lys, Xaa₆ is Glu orgamma-carboxy-Glu (Gla); and the C-terminus contains a carboxyl or amidegroup.
 3. The method of claim 1, wherein the substantially pureτ-conotoxin peptide is modified to contain an O-glycan, an S-glycan oran N-glycan.
 4. The method of claim 2, wherein the substantially pureτ-conotoxin peptide is modified to contain an O-glycan, an S-glycan oran N-glycan.
 5. A method for inducing analgesia in an individual whichcomprises administering an effective amount of a pharmaceuticalcomposition comprising a τ-conotoxin peptide or a pharmaceuticallyacceptable salt thereof and a pharmaceutically acceptable carrier, saidτ-conotoxin peptide having the generic formula I:Xaa₁-Xaa₂-Xaa₃-Xaa₄-Cys-Cys-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Cys-Cys-Xaa₁₀-Xaa₁₁-Xaa₁₂-Xaa₁₃-Xaa₁₄-Xaa₁₅-Xaa₁₆-Xaa₁₇-Xaa₁₈-Xaa₁₉(SEQ ID NO:1), wherein Xaa₁ is des-Xaa₁, Asp, Glu or γ-carboxy-Glu(Gla); Xaa₂ is des-Xaa₂, Gln, Asn, Glu, Trp (D or L), neo-Trp, halo-Trpor any unnatural aromatic amino acid; Xaa₃ is des-Xaa₃, Gly, Ala, Asn orGln; Xaa₄ is des-Xaa₄, Val, Leu (D or L), Ile, Ala, Gly, Glu, Gla, Asp,Ser, Thr, Phe, Trp (D or L), neo-Trp, halo-Trp (D or L) or any unnaturalaromatic amino acid; Xaa₅ is Pro, hydroxy-Pro, Gln, Asn, Glu, Gla, Ala,Gly, Lys, Arg, Ile, Val, homoarginine, ornithine, N-methyl-Lys,N,N-dimethyl-Lys, N,N,N-trimethyl-Lys or any unnatural basic amino acid;Xaa₆ is Val, Phe, Thr, Ser, Glu, Gla, Asp, Asn, Gln, Ala, Gly, Ile, Leu(D or L), Met, Pro, hydroxy-Pro, Arg, homoarginine, ornithine, Lys,N-methyl-Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys, any unnatural basicamino acid or any unnatural aromatic amino acid; Xaa₇ is any Val, Ile,Asn, Leu (D or L), Gln, Gly, Ala, Phe, Glu, Gla, Arg, ornithine,homoarginine, Lys, N-methy-Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys,any unnatural basic amino acid or any unnatural aromatic amino acid;Xaa₁ is Ile, Leu (D or L), Met, Thr, Ser, Pro, hydroxy-Pro, Gln, Asp,Glu, Gla, Asn, Arg, homoarginine, ornithine, Lys, N-methy-Lys,N,N-dimethyl-Lys, N,N,N-trimethyl-Lys, Tyr, nor-Tyr, mono-halo-Tyr,di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr, any unnatural basicamino acid, any unnatural aromatic amino acid or any unnatural hydroxycontaining amino acid; Xaa₉ is des-Xaa₉, Ala, Gly, Asp, Glu, Gla, Trp (Dor L) neo-Trp, halo-Trp (D or L), Lys, N-methy-Lys, N,N-dimethyl-Lys,N,N,N-trimethyl-Lys, Arg, homoarginine, ornithine, Tyr, nor-Tyr,mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr orany unnatural basic amino acid; Xaa₁₀ is des-Xaa₁₀, Ile, Leu (D or L),Val, Glu, Gla, Asp, Thr, Ser, Pro, hydroxy-Pro, Trp (D or L), neo-Trp,halo-Trp (D or L), Phe, any unnatural aromatic amino acid or anyunnatural hydroxy containing amino acid; Xaa₁₁ is des-Xaa₁₁ Gln, Asn,Leu (D or L), Ile, Val, Ala, Gly, Trp (D or L), neo-Trp, halo-Trp (D orL), Arg, homoarginine, ornithine, Lys, N-methy-Lys, N,N-dimethyl-Lys,N,N,N-trimethyl-Lys, any unnatural basic amino acid or any unnaturalaromatic amino acid; Xaa₁₂ is des-Xaa₁₂, Ala, Gly, Phe, Trp (D or L),neo-Trp, halo-Trp (D or L) or any unnatural aromatic amino acid; Xaa₁₃is des-Xaa₁₃, Glu, Gla, Asp, Phe or any unnatural aromatic amino acid;Xaa₁₄ is des-Xaa₁₄, Ile, Val or Leu (D or L); Xaa₁₅ is des-Xaa₁₅, Thr,Ser, Arg, homoarginine, ornithine, Lys, N-methy-Lys, N,N-dimethyl-Lys,N,N,N-trimethyl-Lys or any unnatural basic amino acid; Xaa₁₆ isdes-Xaa₁₆, Glu, Gla or Asp; Xaa₁₇ is des-Xaa₁₇, Asn or Gln; Xaa₁₈ isdes-Xaa₁₈, Asp, Glu or Gla; Xaa₁₉ is des-Xaa₁₉, Phe or any unnaturalaromatic amino acid; and the C-terminus contains a free carboxyl groupor an amide group.
 6. The method of claim 5, wherein the τ-conotoxinpeptide is modified to contain an O-glycan, an S-glycan or an N-glycan.7. A method for inducing analgesia in an individual which comprisesadministering an effective amount of a pharmaceutical compositioncomprising a τ-conotoxin peptide or a pharmaceutically acceptable saltthereof and a pharmaceutically acceptable carrier, said τ-conotoxinpeptide selected from the group consisting of:Phe-Cys-Cys-Xaa₁-Val-Ile-Arg-Xaa₂- (SEQ ID NO:2) Cys-Cys-Xaa₃;Phe-Cys-Cys-Xaa₁-Phe-Ile-Arg-Xaa₂- (SEQ ID NO:3) Cys-Cys-Xaa₃;Cys-Cys-Gln-Thr-Phe-Xaa₂-Xaa₃-Cys- (SEQ ID NO:4) Cys-Gln;Xaa₄-Gly-Xaa₃-Cys-Cys-Xaa₅-Xaa₆-Asn- (SEQ ID NO:5) Ile-Ala-Cys-Cys-Ile;Gly-Cys-Cys-Ala-Arg-Leu-Thr-Cys-Cys- (SEQ ID NO:6) Val;Asn-Gly-Cys-Cys-Xaa₁-Xaa₅-Gln-Met- (SEQ ID NO:7) Arg-Cys-Cys-Thr;Asp-Xaa₃-Asn-Ser-Cys-Cys-Gly-Xaa₅- (SEQ ID NO:8)Asn-Xaa₁-Gly-Cys-Cys-Xaa₁-Xaa₃; Xaa₄-Gly-Xaa₃-Cys-Cys-Xaa₅-Xaa₆-Asn-(SEQ ID NO:9) Ile-Arg-Cys-Cys-Val; Xaa₆-Cys-Cys-Xaa₆-Asp-Gly-Xaa₃-Cys-(SEQ ID NO:10) Cys-Thr-Ala-Ala-Xaa₁-Leu-Thr;Gly-Cys-Cys-Xaa₆-Asp-Gly-Xaa₃-Cys- (SEQ ID NO:11)Cys-Thr-Ala-Ala-Xaa₁-Leu-Thr; Asn-Gly-Cys-Cys-Arg-Ala-Gly-Asp-Cys- (SEQID NO:12) Cys-Ser-Arg-Phe-Xaa₆-Ile-Xaa₅-Xaa₆- Asn-Asp-Phe;Asn-Ala-Cys-Cys-Ile-Val-Arg-Gln-Cys- (SEQ ID NO:13) Cys;Asn-Gly-Cys-Cys-Arg-Ala-Gly-Asp-Cys- (SEQ ID NO:14) Cys-Ser;Cys-Cys-Xaa₁-Arg-Arg-Leu-Ala-Cys- (SEQ ID NO:15) Cys-Ile-Ile;Cys-Cys-Xaa₁-Asn-Xaa₅-Xaa₁-Cys-Cys- (SEQ ID NO:16) Phe-Ile;Gly-Cys-Cys-Ala-Met-Leu-Thr-Cys-Cys- (SEQ ID NO:17) Val;Leu-Cys-Cys-Val-Thr-Xaa₆-Asp-Xaa₃- (SEQ ID NO:18)Cys-Cys-Xaa₆-Xaa₃-Xaa₃; and Val-Cys-Cys-Arg-Xaa₁-Val-Gln-Asp- (SEQ IDNO:19) Cys-Cys-Ser;

wherein Xaa₁ is Pro or hydroxy-Pro; Xaa₂ is Tyr, mono-halo-Tyr,di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr or nitro-Tyr; Xaa₃ is Trp orhalo-Typ; Xaa₄ is Gln or pyro-Glu; Xaa₅ is Lys, N-methyl-Lys,N,N-dimethyl-Lys or N,n,N-trimethyl-Lys, Xaa₆ is Glu orgamma-carboxy-Glu (Gla); and the C-terminus contains a carboxyl or amidegroup.
 8. The method of claim 7, wherein the τ-conotoxin peptide ismodified to contain an O-glycan, an S-glycan or an N-glycan.